Integrated electrokinetic devices and methods of manufacture

ABSTRACT

Devices with electrokinetic elements are disclosed as well as their method of microfabrication for use in micro-scale analysis, mixture separation and reaction. The devices consist of solid hydrophilic-matrix films that have been microfabricated into a variety of micro-scale structures. These structures include hydrophilic-matrix conductors for electrokinetic species transport and separation. They also include hydrophilic-matrix cladding containing chemical species adjacent to either an open conduit or a hydrophilic matrix conductor. Also described are other integrated microstructures consisting of hydrophilic-matrix materials such as micro-reaction zones for retaining chemical species for on-chip chemical reactions and integrated detection structures for on-chip species detection. In general, a hydrophilic matrix on a substrate functions as a conductor that is covered by an electrically insulating, preferably water permeable material.

REFERENCE TO RELATED APPLICATIONS

This Application is a divisional application of Parent Application U.S.Ser. No. 09/871,821, filed Jun. 4, 2001, issued as U.S. Pat. No.7,214,300 entitled Integrated Electrokinetic Devices and Methods ofManufacture.

FIELD OF THE INVENTION

The invention is directed to devices with electrokinetic components forsolute species transport and/or reaction and separation and their methodof manufacture, for use in micro-scale analysis, mixture separation andreaction.

BACKGROUND OF THE INVENTION

Electrokinetic transport (electroosmosis, electrophoresis) of chemicalspecies through thin slabs or through narrow conduits is known in theart. However, more recently, new devices with eletrokinetic-transportelements have been disclosed. In particular, devices described in theliterature have been directed towards applications ofeletrokinetic-transport technology in genomics, proteomics,combinatorial chemistry and high-throughput screening for drugdiscovery.

Electrokinetic-transport technology is used in species separationdevices including slab-gel electrophoresis devices. In the slab-gelelectrophoresis method, separation of chemical species occurs when thespecies in aqueous solution are transported at different rates along thegel. This class of prior-art devices generally consists of macro-scaleslabs of hydrophilic-gel materials. Examples are described in U.S. Pat.No's. 4,574,040 and 4,663,015. In systems such as those described in theabove referenced patents, pouring a gel-forming liquid into the spacebetween two glass plates forms the slabs. The gel-forming liquid is anaqueous solution of hydrophilic polymers and cross-linkers. The gelationprocess causes the liquid to solidify into a solid slab. The resultantgel slab is a solid matrix containing a substantial quantity of water.The slab thickness is determined by the spacing between the platesmaintained by spacer strips placed between and along two opposing edgesof the plates. Clamps hold the plates together and the spacer strips aresmooth so that a seal is formed under the pressure of the clamps,preventing leakage of either the gel-forming solution during gel castingor the buffer solution during electrophoresis.

Methods disclosed to reduce the dimensions of the transport channel ofthe slab-gel devices have generally used macrofabrication techniques.For example, U.S. Pat. No. 5,627,022 discloses a thin gel slab preparedinside a gel holder consisting of two planar substrates and a thinspacer consisting of beads in an adhesive matrix. Microfabricatedmultiple separation lane slab-gel devices have been disclosed, forexample in U.S. Pat. No. 5,543,023. Such devices consist of an array ofthin slabs separated by spacers. Multiple lane devices with gels castinto microchannel arrays are known in the art. U.S. Pat. No. 5,192,412discloses a slab-gel contained within plates wherein one plate has alinear array of microchannels. U.S. Pat. No. 5,746,901 discloses asimilar combination of corrugated and flat glass plates sandwiching gelslabs. U.S. Pat. No. 5,954,931 discloses an electrophoresis device withparallel channels formed by casting gel onto a substrate withmicrochannels. Gel compositions for small dimension electrophoresis gelslabs have been disclosed in U.S. Pat. No. 6,013,166. Macro-scale driedgel slabs that are reconstituted by treatment with water prior to usehave also been reported in the prior art (U.S. Pat. Nos. 4,048,377 and4,999,340).

Species separation devices of the prior art also include capillary tubesused both for capillary electrophoresis and capillary chromatography(for example U.S. Pat. No. 5,207,886). In this technique separations areconducted by electrokinetic flow of liquid through narrow-bore glasscapillary tubes. In these prior-art capillary devices the separationoccurs within the capillary tube and the separation medium is a liquidthat fills the tube after it is introduced through one end of the tube.Some prior-art devices use a polymeric coating on the internal surfaceof the narrow-bore tube (U.S. Pat. Nos. 5,141,612 and 5,167,783), othersuse capillary tubes pre-filled with gel (U.S. Pat. No. 4,997,537), stillothers introduce the separation polymer dissolved in the sample liquid(U.S. Pat. No. 5,089,111).

Multi-lane separation devices consisting of multiple capillary tubesassembled in a housing have been disclosed in the art, for example U.S.Pat. No. 5,439,578. It is well known in the art that such capillaryseparation devices provide superior separation performance over slab-gelseparation devices of the prior art because narrow bores provide forless spreading of the species in the separating medium. Also, because ofsuperior heat dissipation, high voltages can be used to effect rapidseparation.

Some shortcomings of these devices include the inability to easilyintegrate with other fluid manipulation elements or other elements ofthe analytical process and the inability to provide readily forvariations of composition within the medium.

Integrated micro-analytical and micro-chemical-reaction devices,commonly also referred to as lab-on-a-chip devices, have been disclosedin the prior art (for example U.S. Pat. Nos. 4,908,112 5,126,022 and5,180,480). These devices utilize micro-machining methods adapted fromsemiconductor chip manufacturing to fabricate micro or meso-scaledevices on planar substrates for the purpose of performing separations,measurements and chemical reactions. These devices are mechanicalstructures realized by forming cavities and channels or trenches into asolid substrate. The devices are generally completed when a coverassembly over the cavitated substrate provides a cap that converts thecavities and channels into chambers and conduits. U.S. Pat. No.5,429,734 however, discloses a channel etched into a semiconductor waferthat includes a monolithic capping means. U.S. Pat. No. 4,908,112discloses separation devices including electrodes with channels etchedinto semiconductor slabs. U.S. Pat. No. 5,750,015 discloses separationdevices with trenches formed in insulating plastic slabs. Otherstructures consisting of cavitation in planar substrates include deviceswith channels and detectors (U.S. Pat. Nos. 5,637,469 and 5,906,723),devices with chambers (U.S. Pat. No. 5,585,069) and devices withchannels and mechanical sieving means (U.S. Pat. No. 5,304,487).Reactions, mixture separations and analyses take place in suchmicrostructures in liquids that are electrostatically transported alongthe conduits. Generally in these prior art devices, the reactants,catalysts and reagents are stored and prepared in off-chip processesthen introduced into the channels of the chip during use by pumping fromone open end of the channel along its entire length. U.S. Pat. No.5,126,022 discloses microfabricated trenches that are filled with gelprior to use.

Integrated micro-channel separation devices have been disclosed in theart, wherein electrokinetic fluidic manipulations are carried out inmicro-channel structures more complicated than those feasible in asimple capillary tube with only an inlet and an outlet, and morecomplicated than an array of channels either in multi-lane slabs orcapillary tube arrays. U.S. Pat. No 5,770,029 discloses a device with amain electrophoretic channel connected to a secondary, enrichmentchannel. U.S. Pat. No. 5,296,114 discloses an electrophoretic separatingdevice consisting of a channel in the form of a loop with multiple inletand outlet ports. U.S. Pat. No. 5,750,015 discloses a device consistingof a main trench and multiple branching trenches. Devices are disclosedwith multiple connected channels (U.S. Pat. No. 5,800,690), intersectingchannels (U.S. Pat. Nos. 5,599,432 and 6,010,608) and channels connectedto multiple reservoirs (U.S. Pat. No. 5,858,195).

Integrated micro-channel devices in which there is a binding stepcombined with an electrokinetic transport step within a conduit or slabare also known in the art. U.S. Pat. No. 5,661,028 discloses a devicethat integrates a binding/primer element with an element for introducingreagents from off-chip for a Sanger sequencing reaction with anelectrophoretic separation element consisting of a planar etched channelwith glass cover plate backfilled with gel. U.S. Pat. Nos. 4,628,035 and5,055,415 disclose antigen-antibody binding inside an electrophoreticmedium.

The prior art of biosensors and dry reagent diagnostic devices containsnumerous uses of hydrophilic materials or gels. Devices from this priorart that are made by microfabrication include for example U.S. Pat. No.5,194,133 that discloses a biosensor with a micromachined channel filledwith a gel material. Devices that consist of a composite of agas-permeable layer and a hydrophilic-polymer layer also are known inthe prior art of biosensors, including devices of this type made bymicrofabrication. For example U.S. Pat. No. 4,933,048 discloses amicrofabricated gel and hydrophobic-vapor-permeable polymer for use as asalt bridge of a potentiometric reference electrode. U.S. Pat. Nos.5,514,253 and 5,200,051 disclose micro-fabricated gas and enzymebiosensors that also utilize these composite layers. These numerousdiagnostic devices disclosed in the prior art of biosensors utilize thegel or hydrophilic material as a medium for reagent retention or as anelement through which species move by diffusion. However, none of thesereferences teach the use of a layer composite of this general type in anactive electrokinetic pumping application. Both the functional designand the mode of operation of this class of prior-art biosensor anddry-reagent diagnostic devices are different from active electrokineticpumping devices.

Devices have been disclosed in the prior art that utilize voltages notfor electrokinetic transport but for modulating the amount ofhybridization at an electrode surface (U.S. Pat. Nos. 5,632,957 and6,017,696) or for biological sample preparation (U.S. Pat. No.6,129,828).

In summary, prior-art electrokinetic devices are either empty channels(trenches in planar substrates or conduits in tubes), channels withcoated surfaces, or channels filled with polymer solutions or gel.Prior-art devices also include slabs of gels or gel tracks formed bycasting gels into mechanical pre-forms or cavities. The prior-artdevices are thus limited in one of several ways. Prior-art micro-channeldevices, while manufactured in part by microfabrication methodologies,generally only provide for elements that contain mechanical structures.Thus they do not contain the chemicals and reagents required to functionas truly integrated-analytical systems. At the current state of the artthese types of devices consist of really only lab-glassware-on-a-chiprather than the complete lab-on-a-chip as they have been called. Theprior art does not teach how the integration of chemical function can beaccomplished with any generality. Furthermore, prior-art slab-gel baseddevices are generally made by traditional macro-fabrication methods,thus they are expensive to manufacture and use. They require largesample sizes and are slow in performance. They cannot easily beintegrated either to provide multi-analysis capability, nor easily orcost effectively be combined with other components of an integratedanalytical system.

Moreover, the materials of the transport element of prior-art slab-geldevices have been limited to gelatinous media. As such they are largelywater-based and fragile and difficult to process into structures muchmore complicated than simple slabs. These materials are not amenable toplanar processing nor microfabrication to make integrated devices. Thusthere remains a significant need for cost-effective electrokineticdevices amenable to planar processing and/or microfabrication and forprocesses for their manufacture. A further need exists forelectrokinetic devices with incorporated chemical entities.

SUMMARY OF THE INVENTION

It is an object of the invention to provide electrokinetic devices and amethod of fabrication therefor, which devices are preferably applicablefor use in micro-scale analysis, mixture separation and reaction.

It is yet a further object of the invention to provide electrokineticdevices including hydrophilic-matrix conductors for electrokineticsolute species transport and/or separation.

It is another object of the invention to provide electrokinetic devicesthat are suitable for planar processing and/or microfabrication.

It is still another object of this invention to teach methods wherebyhydrophilic matrixes and included chemicals may be microfabricated.

It is a further object of the invention to teach methods wherebyencapsulating elements can be microfabricated.

It is still a further object of the invention to provide electrokineticdevices featuring the integration of hydrophilic-matrix conductors withchemical entities contained in reservoirs and reaction regions toprovide for self-contained micro-analytical systems. Chemical entitiesinclude separation polymers, attachment ligands or probes, primers,enzymes, filtration means, buffers and the like.

It is yet another object of the invention to provide devices thatinclude self-contained reagents and are formatted as a cost-effective,single-use disposable device for use particularly in the fields ofgenomics and proteomics and molecular diagnostics.

Complex embodiments of devices in accordance with the present inventionwill in the following be collectively called integrated-electrokineticcircuits.

It is an object of the invention to provide integrated-electrokineticcircuits and their methods of manufacture.

It is an object of this invention to teach microfabrication methods forintegrated-electrokinetic circuits that retain their chemicals duringback-end processing steps. These objects of the invention are achievedin a device for electrokinetic transport of an aqueous solute, includingan electrically insulating substrate; an electrokinetic conductorelement in the form of a solid hydrophilic-matrix layer on thesubstrate, the matrix layer being in a substantially dry, inactive stateand having a first surface engaging the substrate and a second surface;and a cover layer for electrically insulating and covering the secondsurface, the cover layer being impermeable to the solute; wherebyexposure of the hydrophilic matrix to water converts the matrix from theinactive state to a hydrated, active state permitting electrokinetictransport of the solute.

In a preferred embodiment, the solid hydrophilic-matrix layer is in theform of a film that can be fabricated into a variety of micro-scalestructures. The solid hydrophilic-matrix layer when hydrated functionsas a conductor for electrokinetic species transport or separation.

In another embodiment, the device is manufactured in the form of a chipand further includes hydrophilic-matrix cladding containing chemicalspecies adjacent to either an open conduit or a hydrophilic-matrixconductor, as well as other integrated microstructures for retainingchemical species for on-chip chemical reactions and integrated detectionstructures for on-chip species detection.

In devices in accordance with the invention, the transport of species,reactions, mixture separations and analyses takes place withinhydrophilic-matrix conductors, within hydrophilic-matrix sheathedconduits, as well as within other formed elements such as reservoirs andreaction zones that consist of solid-state hydrophilic matrices intowhich water is introduced at or before the point of use.

Although reference is made throughout this application to transportationof a solute species, this term is intended to encompass transport of thesolute species irrespective whether the solute is transported within thesolvent or by way of a pumping of the solvent, for example byelectroosmosis.

In another preferred embodiment of this invention, elements ofintegrated-electrokinetic circuits are produced by microfabrication.Thus, for example a particular circuit component of anintegrated-electrokinetic circuit in accordance with this invention is aconductor for transport of a solute chemical species. The conductorpreferably consists of a thin film of a solid hydrophilic-matrixmaterial that has been patterned into a strip-line by a microfabricationmethod. This microfabrication is preferably performed on the hydrophilicsolid in its dry or semi-dry form. The thin film is preferably less than10 micrometers in thickness. Preferred dimensions of the strip-line areless than 100 micrometers in width and greater than 100 micrometers inlength. In the preferred circuit, the solid hydrophilic-matrix conductoroverlays a substantially planar substrate that is impermeable to thesolute species to be transported through the hydrophilic conduit. Theconductor is surrounded on its sides and top by a cover layer made ofencapsulant materials also substantially impermeable to the transportedspecies. In the preferred embodiment the encapsulant material isdeposited by a film process and also is formed by microfabrication.

In still a further preferred embodiment of the invention, at least aportion of the insulating encapsulant material has the additionalproperty that it is permeable to water vapor. This allows for theinitially substantially dry and inactive hydrophilic matrices to rapidlytake up water being transported through the encapsulant material asvapor. Upon exposure to water, the encapsulant retains the insulatingproperties that are required for the proper function of the device. Thedry solid hydrophilic matrix however becomes a conducting electrolyteupon incorporation of water. The device is exposed to water eitherbefore or at the point of use of the device. In a variant of thispreferred embodiment it is the substrate material that is waterpermeable.

In another embodiment of this invention, integrated-electrokineticcircuit components are provided where hydrophilic-matrix conductors,sheaths, reservoirs and reaction zones are prepared with in-situchemical reagents for performing reactions, mixture separations oranalyses. These in-situ chemicals are preferably introduced into thehydrophilic matrix at manufacture.

In still another embodiment, the device includes hydrophilic-matrixconductors in parallel arrays to facilitate transport of solute speciesthrough multiple lanes as might be used in a multiple sample separationon a single integrated device, or as might be used to transportchemicals to multiple reaction zones or multiple regions ofligand-binding. In yet an additional embodiment, the device includeshydrophilic-matrix conductors with intersections so as to facilitatemovement of species from one conductor to another according to the timedapplication of voltages across the conductors.

In another embodiment, the device includes hydrophilic-matrix conductorsthat intersect but are isolated one from another to prevent electricalor solute species contact.

In a preferred embodiment the device includes hydrophilic-matrixconductors with integral electrodes. Thus for example, a particularcircuit component of an integrated-electrokinetic circuit in accordancewith the invention is an encapsulated hydrophilic-matrix conductor withintegral electrodes for electrokinetic species transport. This circuitcomponent can serve also as a column element in a separation device. Thepreferred device consists of a microfabricated hydrophilic-matrixconductor and microfabricated cover layer of encapsulant material.

The hydrophilic-matrix conductor preferably has one end through which asample to be transported or separated can be introduced, and another endwhere the transported fluid flows out. The conductor is preferablydisposed over microfabricated electrodes that provide the electromotivedriving force to cause electrophoretic transport of charged species orelectroosmotic flow of solvent. Solute species separation in the deviceof the invention occurs because of differential mobility of transportedions (as occurs in conventional slab-gel or capillary electrophoresis),or by differential residence at absorptive sites within a column element(as in conventional chromatography methods).

In a further embodiment of this invention, the device includeshydrophilic-matrix conductors with variable chemical composition alongtheir length. In this embodiment, a hydrophilic-matrix conductor has afirst region of a hydrophilic matrix interposed between two electrodesthat provide the electrokinetic driving force. A second and third regionare upstream and downstream of the first hydrophilic-matrix region. Thehydrophilic matrix of the first region is composed of a material of highelectroosmotic coefficient to maximize flow rate at a given appliedvoltage. The composition of the second and third regions is chosen tooptimize for some other functional characteristic. For example acomposition appropriate to perform a binding reaction, a separation or aspecies detection. Another example of this aspect of the invention is ahydrophilic-matrix separation element with graded pore size along itslength.

In yet another preferred embodiment of this invention a reaction zoneincorporating chemical entities for reaction is integrated with ahydrophilic-matrix conductor. A particular circuit component of theintegrated-electrokinetic circuit is a reaction zone with inlet andoutlet ports and means for transporting reactant chemicals and productsrespectively to and from the reaction zone.

In another embodiment of this invention, an integrated-electrokineticcircuit includes reagent reservoirs and waste reservoirs.

In still another embodiment of this invention, anintegrated-electrokinetic circuit includes integral detectors, mostpreferably electrochemical detectors.

In a further embodiment of this invention a micro-analytical systemfeatures an integrated-electrokinetic circuit consisting of a number ofdifferent hydrophilic-matrix components. These includehydrophilic-matrix conductors, reservoirs, electrokinetic pumps,conductor junctions, integral electrodes, reaction zones and detectors.It is possible to perform numerous micro-analytical procedures using thedevice according to this invention. These procedures includeligand-binding assays, separations, PCR or primer extension reactions,as well as methods employing combinations of reactions and/orseparations.

In another embodiment of this invention a micro-analytical systemfeatures a ligand-binding array with each binding element connected toan integrated electrokinetic pump consisting of hydrophilic-matrixconductors and integral electrodes. Target molecules in a samplesolution are electrostatically pumped to a binding region as they passthrough an orifice into the hydrophilic-matrix conductor. In this waythe forced-convective flow of sample solution effects rapid speciestransport to the binding molecules within the binding layer. This deviceprovides enhanced speed of response as well as better sensitivitycompared to conventional ligand-binding arrays on non-porous substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further discussed in detail by way of exampleonly and with reference to the following drawings, wherein:

FIG. 1A is a schematic perspective view of a hydrophilic-matrixconductor according to a preferred embodiment of the invention;

FIG. 1B shows a schematic top plan view of the hydrophilic-matrixconductor of FIG. 1A;

FIG. 1C is a horizontal cross-section through the embodiment of FIG. 1Btaken along line B-B′;

FIG. 1D is a horizontal cross-section through the embodiment of FIG. 1Btaken along line A-A′;

FIG. 1E is a horizontal cross-section through the embodiment of FIG. 1Btaken along line B-B′;

FIG. 2 is a diagram of various prior art devices;

FIG. 3A is a schematic perspective view of a hydrophilic-matrixconductor with solvent insoluble termini according to another embodimentof the invention;

FIG. 3B shows a schematic top plan view of the embodiment of FIG. 3A;

FIG. 3C is a horizontal cross-section through the embodiment of FIG. 3Btaken along line B-B′;

FIG. 3D is a horizontal cross-section through the embodiment of FIG. 3Btaken along line A-A′;

FIG. 3E is a schematic perspective view of a hydrophilic-matrixconductor with hydrophilic-matrix sheath according to an embodiment ofthe invention;

FIG. 4A shows a schematic perspective view of a hydrophilic matrixconductor with integrated electrodes according to a further preferredembodiment of the invention;

FIG. 4B is a schematic top plan view of the embodiment of FIG. 4A;

FIG. 4C is a horizontal cross-section through the embodiment of FIG. 4Btaken along line C-C′;

FIG. 4D is a horizontal cross-section through the embodiment of FIG. 4Btaken along line A-A′;

FIG. 4E is a horizontal cross-section through the embodiment of FIG. 4Btaken along line B-B′;

FIG. 5A is a schematic perspective view of an integrated-electrokineticcircuit according to an embodiment of the invention;

FIG. 5B is a schematic top plan view of the embodiment of FIG. 5A;

FIG. 5C is a horizontal cross-section through the embodiment of FIG. 5Btaken along line C-C′;

FIG. 5D is a horizontal cross-section through the embodiment of FIG. 5Btaken along line A-A′;

FIG. 5E is a horizontal cross-section through the embodiment of FIG. 5Btaken along line B-B′;

FIG. 6A is a schematic top plan view of an array of hydrophilic matrixconductors with integral electrodes and an array of reaction zones ateach input orifice of the array of conductor elements, according toanother embodiment of the invention;

FIG. 6B is a horizontal cross-section through the embodiment of FIG. 6Ataken along line B-B′;

FIG. 6C is a horizontal cross-section through the embodiment of FIG.6Ataken along line A-A′;

FIG. 6D is a horizontal cross-section through the embodiment of FIG.6Ataken along line B-B′;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Throughout the following description, equivalent elements are referredto by the same reference numbers.

FIGS. 1A and 1B are respectively a perspective view and a plan view of afirst embodiment of the invention. Cross-sections through thisembodiment are shown in FIGS. 1C and D. FIG. 2 shows for comparisonelectrokinetic-transport devices of the prior art.

The device of FIGS. 1A and 1B consists of a planar insulating solidsubstrate 1, and an overlayer of a solid hydrophilic-matrix material 2.Matrix layer 2 is spatially defined as a strip-line with a longitudinaldimension and provides an electrolyte conductor along which chemicalsolute species can be transported. The conductor 2 is covered by a coverlayer 3 of a water vapor-permeable material and has openings 4 a and 4 bthat allow the entry and exit of solute species to be transportedthrough the conductor. Openings 4 a and 4 b are shown as located ateither end of conductor 2. Also possible are passages through the waterpermeable layer, as shown by 4 a′ and 4 b′ in FIG. 1. Also possible areopenings through the side or lower surfaces. The conductor 2 isconnected to other elements of the device through openings 4 a and 4 b.Thus in some uses of the conductor of this embodiment it may beconnected to a test solution at one end through 4 a and a reservoir atthe other through 4 b. Solute species can be electrostatically pumpedthrough 4 a from the test solution into the conductor, then along theconductor to the reservoir through 4 b. In other uses of the conductorof this embodiment 4 a and 4 b may be connected to otherhydrophilic-matrix conductors or to other microfabricated elements suchas reaction zones, reservoirs and the like as part of anintegrated-electrokinetic circuit.

Referring to the B-B′ cross-section of FIG. 1C, in operation a portionof the top surface of the device is immersed in an aqueous medium. Water50 is transported as its vapor through the water vapor-permeable coverlayer 3, into the initially dry, inactive hydrophilic solid matrix 2.The cover layer 3 is otherwise insulating i.e. it does not transportother solute species, ions or electrons. After water has been absorbedinto the matrix 2, it is rendered functionally equivalent to an aqueouselectrolyte. Species 51 that are introduced into the wire throughopening 4 a, or dry solid reagent initially present in the hydrophilicmatrix at manufacture, can move through the wire by electrokinetictransport through conductor 2 when excited by application of a voltage.Species 52 exit the conductor 2 at opening 4 b. The voltage is appliedby two spaced apart electrodes (not shown) that make electrical contactto the hydrophilic-matrix layer. Such electrical contact means are wellknown to those skilled in the art. They include electrodes in the formof contacting pins that are brought into electrical contact with theelectrokinetic-conductor layer on the substrate, as well as integralelectrodes described further below.

The specific composition of the material of layer 2 depends on itsfunction in the device. The primary function of the hydrophilic-matrixmaterial is to provide for a physical support structure into which watercan be incorporated to render the layer functionally an aqueouselectrolyte. In separation applications described in later embodimentsof this invention, the conductor is used as a separation column. Inthese applications the composition of the conductor layer 2, is selectedto provide optimum separating properties that are determined by theshape and size of the species to be separated. In applications where ahigh flow rate of solution through the conductor is the importantproperty then the composition is chosen to maximize the electroosmoticcoefficient.

The hydrophilic-matrix 2 consists of a material composed of eithermonomeric or polymeric hydrophilic molecules that readily incorporatewater. Examples are sugars, starches, alcohols, ethers, poly amino acidsproteins and hydrophilic silanes and derivitized silanes. Thehydrophilic matrix 2 may consist of a hydrophilic polymer in an extendedstate such as in a gel. Absorption of water results in a gel-likepolymer in which water is incorporated into polymer chain interstices.Examples of suitable materials are cross-linked polyvinyl alcohols, polyhydroxy methacrylates, polyacrylamides, agarose, gelatins and silanes.The hydrophilic matrix 2 may be formed from a latex. The hydrophilicmatrix may also contain dry electrolyte salts (to achieve high internalosmolality for good water uptake), buffers (to regulate internal pH forcontrol of swelling of the hydrophilic matrix and to regulate internalpH to control chemical species transport and reaction) and otherreagents depending on the function of the device in which the conductoris used.

The water vapor-permeable layer may be manufactured from a variety ofdifferent materials. Low density, hydrophobic hydrocarbon andfluorocarbon polymers are insulating and water permeable. Silicones,siloxanes, silicone-polycarbonate copolymers are preferred materialsbecause they are insulating and highly water vapor permeable. The mostpreferred materials are dimethyl polysiloxane and silicone polycarbonatebecause they can endure a significant physical expansion of theunderlying material as water is absorbed.

The device of the embodiment of FIG. 1 is fabricated usingmicrofabrication technology. For example the substrate 1 may be anyplanar material suitable for use in microfabrication equipment such assilicon, a ceramic, a glass or a polymer. The substrate material can beinsulating as is, or it can be coated with a material to render itinsulating. For example, if the substrate is semiconducting silicon itcan be coated with insulating silicon dioxide according to numeroustechniques well known in the art. The substrate may be partiallymanufactured, in which case it already supports microfabricated conduitsand chambers with insulating coatings, and a next level ofelectrokinetic circuitry is being fabricated. Some methods ofmicrofabricating hydrophilic solid matrix layers that are appropriate totheir use in biosensors are well known in the prior art of biosensors.Compositions and methods which can be used for microfabricatinghydrophilic-matrix layers for the electrokinetic devices of thisinvention are not known in the art

In one embodiment of the manufacturing method of the current invention,photo-formable formulations are used for the manufacture of thehydrophilic matrix. Additives to hydrophilic polymer materials thatcause cross-linking upon exposure to radiation are well known. Suchadditives when formulated with the other components of the hydrophilicmatrix in accordance with the invention render the cast polymer filmphoto-formable. The process of photo-forming is similar to theprocessing of a standard photoresist. A layer of the material isdeposited on a planar substrate by spinning, spray printing, dipping orcasting. It is allowed to dry. The dried layer is exposed to actinicradiation through a mask. UV exposure is common, although otherwavelengths of light are possible depending on the additive component'swavelength sensitivity. Electron beam formable materials also arefeasible. The exposed film is then developed in a developing medium in abath, or spray or even a dry plasma process. For the wet developmentprocesses aqueous developing solutions are typically used. It is adisadvantage of such a process that salts and other chemicals that mightbe necessary for the proper operation of the device would be removedfrom the hydrophilic matrix during the wet development process. Anotherdisadvantage of photo-formable layers is the potential deletereouseffect on the intended properties of the final hydrophilic matrix of thephoto-active additives.

A preferred embodiment of the manufacturing method, a more generalapproach to photo-forming hydrophilic-matrix layers, is disclosed here.This process is particularly suited to processing of hydrophilic-matrixmaterials containing salts and other dissolvable reagents for theelectrokinetic-conductor applications of the current invention. Theprocess utilizes completely dry plasma etching steps on hydrophilicmaterials designed to be ash free when plasma etched.

By way of example, a hydrophilic-matrix material containing electrolytesalts and buffers is deposited on a planar substrate from an aqueoussolution by spinning, spraying, printing or dipping. Spinning ispreferred. A photoresist layer is coated from a non-aqueous solvent overthis. It is exposed and developed. The photoresist pattern is thentransferred by etching into the underlying hydrophilic-matrix materialusing a plasma process that leaves no ash in the etched areas. Theplasma etch step concurrently removes the photoresist layer. Forexample, when the hydrophilic matrix contains only carbon, hydrogen,oxygen and nitrogen an oxygen plasma will etch the material forming onlyvolatile etch products and no ash. In this example the hydrophilicmatrix should be formulated with non-metallic salts and buffers to beash-free during oxygen plasma etching. Thus the preferred compositionsof the hydrophilic matrix and its electrolytes, buffers and reagents arethose suitable for ash-free plasma etch processing. Using the abovedescribed ash-free dry processing techniques one or more hydrophiliclayers may be sequentially processed into formed structures withoutexposure to wet developers. All of the components of the films areretained during the process. No potentially deleterious additives arerequired.

The water vapor permeable cover layer 3 may be deposited from the vaporphase using techniques well known in the microfabrication art such assputtering, plasma deposition, or glow discharge polymerization.Preferably however, the water vapor permeable layer is deposited fromsolution. Solvent-castable material compositions such as silicones,siloxanes or silicone polycarbonates are thus preferred. The cover layer3 is preferably photo-formed or patterned using a standardphotolithography and subtractive etching method.

Those skilled in the art will appreciate that devices in which there israpid water absorption into the dry hydrophilic matrix could also befabricated by making the insulating substrate 1, or an insulatingcoating on it, out of a water-permeable material. In general, deviceswith any suitable arrangement of a water-permeable insulating materialin contact with at least a portion of the dry hydrophilic matrix willfacilitate wet-up of the hydrophilic matrix.

It will be apparent to those skilled in the art of lab-on-a-chip devicesthat it is possible to configure the single conducting element of theembodiment described in FIG. 1 as part of a more complexintegrated-electrokinetic circuit. It is well known to practitioners ofintegrated-circuit technology that the methods of planarmicrofabrication such as those disclosed for the fabrication of theembodiment of FIG. 1 are particularly suited to the fabrication of morecomplex structures such as the integrated-electrokinetic circuitsdisclosed herein. The conductor of the embodiment of FIG. 1 may be acomponent of a device consisting of an array of such conductorsintegrated on a single planar substrate. Such anintegrated-electrokinetic circuit will be useful in numerousmicro-chemistry applications. The array can be used to perform numerousseparations at the same time, or the it can be used to transportnumerous micro-batches of test solutions to arrays of reaction zones toperform multi-analyte assays at the same time. Another example is anintegrated-electrokinetic circuit having intersecting and contactingconductor elements so that aliquots of solution being transported downone conductor can be electrostatically transferred to another. Evencomplex conductor geometries of these types of devices can be readilyfabricated through a single photo-process step of the hydrophilic-matrixconductor layer, a so-called single-level process. Anintegrated-electrokinetic circuit can also consist of conductor elementsthat intersect but are isolated one from another. In this example of atwo-level conductor process, a first hydrophilic-matrix layer is formedinto a conductor element by photolithography. The conductor is coatedwith an insulating layer, photo-processed to cover the first hydrophilicmatrix and to form openings, then a second hydrophilic-matrix layer isformed and photo-processed into a conductor element that crosses thefirst conductor but is separated from it by the insulator. Finally thereis a second insulator coating on the second conductor level. Furtherexamples of integrated-electrokinetic circuits are described in theembodiments of FIGS. 5 and 6.

FIG. 2 shows schematics of prior art electrokinetic devices. As wasdiscussed in the background to this invention, there are threecategories of prior-art devices. Schematic 100 shows some of thefunctional components and general dimensions of prior art slab-geldevices. In contrast to the devices of this invention they are producedby macro-fabrication methods. The devices are prepared by casting a gelslab 101 onto a plastic or glass support 102. Prior-art devices such asthese are presented for use with the gel in its wet form.

Schematic 110 shows the general form and typical dimensions of aprior-art capillary tube used for capillary electrophoresis. The priorart capillary tubes are glass pipes with circular cross-section.

Schematic 120 shows the general form and typical dimensions of planarmicro-channel devices of the prior art. Capillary sized channels 122 areformed into planar slabs of insulator 121 and capped with an insulatingcover 123. The resulting cavity closely emulates the cross-sectionaldimensions of the prior-art capillary tube.

In contrast to the devices of the current invention, devices 110 and 120of the prior art are empty pipes or channels into which electrolyte isintroduced at the point of use through one of the open ends of the pipeand the capillary is filled by pumping of the fluid along the length ofthe pipe prior to the electrophoretic separation.

FIGS. 3A-D show a further embodiment of the invention. The Figuresrespectively illustrate a perspective view and a plan view withhorizontal cross-sections A-A′ and B-B′ of a conductor consisting of ahydrophilic-matrix conductor with insoluble termini. In FIGS. 3A-D thedevice consists of a planar, insulating solid substrate 1, an overlayerof a hydrophilic-matrix material 2. Hydrophilic-matrix layer 2 isspatially defined as a strip-line with a longitudinal dimension alongwhich species can be transported. It is delimited at either end byinsoluble termini 5 a and 5 b that are made of a hydrophilic-matrixmaterial that does not dissolve in water. The cover layer 3 of watervapor-permeable material includes openings 4 a and 4 b that allow theentry and exit of solute species to be transported through theconductor. Openings 4 a and 4 b are located at the position of thetermini 5 a and 5 b.

Referring to the B-B′ cross-section of FIG. 3C, in operation a portionof the top surface of the device is immersed in an aqueous medium. Water50 is transported as vapor through the water vapor-permeable cover layer3 into the initially dry, inactive hydrophilic-matrix 2 (as in the caseof the FIG. 1 embodiment). The cover layer 3 is otherwise insulating,that is, it does not transport other solute species, ions or electrons.The terminal regions 5 a and 5 b which are intended to come into directcontact with aqueous media (either during use of the device or duringback-end manufacturing process steps) provide a barrier so that contentsof the hydrophilic-matrix material 2 do not dissolve out into theaqueous media. For example, layer 2 may contain polyethylene glycol.Then, termini 5 a and 5 b may be made of a cross-linked polymer that isimpermeable to polyethylene glycol. The function of the termini 5 a and5 b is to allow electrokinetic transport into the conductor 2 ofselected species in the aqueous medium 51 (solute for transport to areaction, species to be separated or detected) through opening 4 a whilepreventing efflux of larger molecules that are fabricated into thematrix of conductor layer 2. These molecules are retained within theconductor layer 2 during the course of the device's operation. Species52 exit the conductor through opening 4 b.

The embodiments of FIG. 3A-3D (as well as the embodiments of FIG. 3E andFIGS. 5 and 6) shows examples of devices with hydrophilic-matrix layersformed with different compositional regions. Those skilled in the artwill recognize that the ability to fabricate electrokinetic conductorswith regional compositional variations could be advantageous in otherapplications of these devices. Practitioners of the art will alsorecognize that advantageous compositional variations of theelectrokinetic transport medium are difficult to realize in the bulkcast slab-gel devices or in the empty capillary pipe or channel devicesthat are filled with transport medium by pumping along the length of thepipe or channel at the point of use. One example of the prior art whereregional compositional variation has been achieved is the macro gel-slabused in prior-art two-dimensional protein separation devices. The macrogel-slab is fabricated with its composition varying in a directionorthogonal to the electrokinetic transport direction. This compositionalvariation is designed to effect a spatially dependent pH orthogonal tothe transport direction. Such materials are suited to two-dimensionalseparation of proteins by virtue of the pH dependence of the proteinmolecules'charge and hence electrophoretic mobility. A micro-scaledevice of this type or an integrated array of such devices can befabricated using the technology of the current invention.

In another example, the hydrophilic-matrix conductor has a regionalvariation of pore size along the transport direction. This property willcause there to be regional variation of electrokinetic mobility oftransported species. This is advantageous in a separation device wherethe species to be separated have a wide range of mobilities. In auniform-pore separation medium, high molecular weight molecules willneither be transported far nor well separated in the time it takes smallmolecules to traverse the full length of the medium. In a graded-poredevice these differences will be reduced. Such a compositional variationcan be effected readily using microfabrication technology. In onetechnique a hydrophilic matrix layer is formulated with radiationinduced cross-linkers as in a standard photoresist. The degree ofcross-linking is dependent on the radiation dose, also as in a standardphotoresist. Different regions of the layer when exposed to differentdegrees will give differently cross-linked regions. Less cross-linkedregions will have larger pores and higher mobilities, highlycross-linked regions smaller pores and lower mobilities.

In some uses of hydrophilic-matrix conductors according to thisinvention it might be advantageous to provide for a rapid conductingpath in species contact with an adjacent material such that species caninter-diffuse between them. Such a device is shown in FIG. 3E. It isstill another example of a device with compositional variation of itshydrophilic-matrix materials. There is a microfabricatedhydrophilic-matrix conductor 2 with a cladding layer of a secondhydrophilic-matrix material 5 running along its length and in contactwith it. Both hydrophilic-matrix layers are coated with awater-permeable insulating cover layer 3. As in the FIG. 3A example,electrokinetic transport of species is primarily along conductor 2.Hydrophilic matrix 5 may contain absorption sites for retention ofspecies traversing the conductor 2 during the use of the device in anelectrokinetic chromatographic separation application. Hydrophilicmatrix 5 may contain reagents (salts, buffers or polymers and the like)that, when released into conductor 2, regulate the transport propertiesthrough the conductor. In the FIG. 3E embodiment, the cladding layer 5is first formed on substrates, then conductor 2 is deposited onto it andmicrofabricated to be co-linear with 5. There are clearly other spatialarrangements of the two hydrophilic matrix layers that will provideequivalent function as the embodiment of FIG. 3E. For example, it ispossible to first microfabricate layer 2 and then layer 5 so that layer5 is on top of layer 2.

FIGS. 4A-E show another embodiment of the invention. The Figuresrespectively illustrate a perspective view and a plan view withhorizontal cross-sections A-A′ and B-B′ and C-C′ of a hydrophilic-matrixconductor with integral electrodes. In FIGS. 4A and 4B the deviceconsists of a planar, insulating solid substrate 1 with two electrodes 7a and 7 b spaced apart on the surface of the substrate. Electrodes 7 aand 7 b are electrically insulated by layers 8 a and 8 b along thelength of the electrodes. The conductor 2 is in the form of an overlayerof a hydrophilic-matrix material applied to the substrate and spatiallydefined as a strip-line with a longitudinal dimension along whichspecies can be transported. Spaced apart portions of the conductor layer2 are located over the spaced apart electrodes 7 a, 7 b so that theelectrodes are at different positions along the long dimension of thelayer 2. Passages through the insulators 8 a and 8 b are provided atlocations 9 a and 9 b that permit electrical contact between electrodes7 a and 7 b and the conductor layer 2. In use, the other ends ofelectrodes 7 a and 7 b (not shown in the diagrams) are connected toelectrical circuits that supply electrical power to the electrodes. Acover layer 3 of a water vapor-permeable material is applied over theconductor layer 2. There are openings 4 a and 4 b that allow the entryand exit of species to be transported through the conductor.

Referring to the C-C′ cross-section of FIG. 4C, in operation a portionof the top surface of the device is immersed in an aqueous medium. As inthe case of the embodiment of FIG. 1, water 50 is transported as itsvapor through the water vapor permeable layer 3 into the, initially dry,inactive hydrophilic solid matrix 2. The cover layer 3 is otherwiseinsulating, that is, it does not transport other solute species, ions orelectrons. These molecules are retained within conductor layer 2 duringthe course of the device's operation. After water has been absorbed intothe matrix 2, it is rendered functionally equivalent to an aqueouselectrolyte. Species 51 introduced into the conductor layer throughopening 4 a, or dry solid reagent initially present in the hydrophilicmatrix at manufacture, become transportable by convective flow of theelectrolyte within conductor layer 2 when electrostatically pumped, orwhen the solutes are electrically charged, by electrophoretic transportin an electric field. The application of an electrical potentialdifference between electrodes 7 a and 7 b provides the electromotiveforce for electrokinetic transport of species. Positively chargedspecies 54 will drift towards the cathode, negatively charged species 55towards the anode. Also, the entire aqueous electrolyte within thehydrated hydrophilic matrix will be pumped by electroosmosis. As isknown in the art, the zeta potential of static surfaces within the solidmatrix and its walls is generally negative and the flow of the body ofelectrolyte 53 within the matrix 2 will be toward the negative electrodeas shown in FIG. 4C.

Electrodes 7, insulators 8 and passages 9 are manufactured by standardmicro-fabrication methods. Preferably the compositions and methods ofmanufacture of these structures are taken directly from standardprocesses employed in high volume manufacture of silicon chips. Thuselectrodes 7 are polysilicon or refractory metal, or refractory metalsuicides or gold, for example. Insulator 8 is silicon dioxide orpolyimide, for example.

Those skilled in the art will recognize that there are other possiblearrangements of integral electrodes for connection to thehydrophilic-matrix conductor. For example, a device utilizing asubstrate with electrodes on the opposite side to the hydrophilic matrixconductor, having holes through the substrate to provide electricalcontact between electrodes and the conductor will function equivalentlyto the embodiment shown in FIGS. 4A-4E.

The device of FIGS. 5A-5E is an example of an integrated-electrokineticcircuit featuring hydrophilic-matrix conductors and integral electrodes.This device is a self-contained micro-analytical system on a chip. Inaddition to hydrophilic matrix conductors taught in the embodiments ofFIGS. 1-3 and an integrated electrokinetic pump taught in the embodimentof FIG. 4, this embodiment teaches additional integrated-electrokineticcircuit elements such as reservoirs, reservoirs containing reagents,conductor junctions, reaction zones and integral probe-electrodes. Inthis embodiment there is a first hydrophilic-matrix conductor 2, and asecond, optional hydrophilic-matrix conductor 2 d contacting it andforming a junction with it. Conductors 2 and 2 d are in the form ofstrip-lines. There is a hydrophilic-matrix reservoir 2 a contactingconductor 2 at one end and a second reservoir 2 b contacting conductor 2at the other end. Optional hydrophilic-matrix reservoir 2 c contactsoptional conductor 2 d at its end. Reservoirs 2 a, 2 b and 2 c have alarge surface area and volume relative to conductors 2 and 2 d. Thereservoirs can contain dry reagent when fabricated, as determined by thespecific application of the micro-analytical system. There are integralelectrodes 7 a, 7 b and 7 c providing electrical contact to each of thereservoirs. Electrodes 7 are insulated along their length with insulator8. Passages through the insulators 8 a, 8 b and 8 c are provided atlocations 9 a, 9 b and 9 c that permit electrical contact betweenelectrodes 7 a and 7 b and 7 c and the hydrophilic matrix reservoirs 2a, 2 b and 2 c. All of the above hydrophilic-matrix circuit componentsare coated with an insulating but water-permeable cover layer 3. Thereis an opening 4 through cover layer 3 over conductor 2.

The device of this embodiment also constitutes a micro-electrokineticpumping system for delivery of fluids through conductor 2. Fluids may bepumped from reagent reservoirs 2 a or optional 2 c, along conductor 2 toa region 6 of the device where there are separators, analytical cells orreactors as described below, then to a waste reservoir 2 b. Reactionzone 6, including a means for monitoring species concentration therein,is a hydrophilic-matrix region along conductor 2 in which chemicalreactions, separations and species detections take place. Theconcentration of chemical species within the reaction zone 6 may beprobed by a variety of techniques well known in the art including byoptical absorbance, by luminescence or laser induced fluorescence ofluminescent or fluorescent molecules or labels in the reaction zone 6 byoptical detector 11 (not illustrated) or by electrochemical detectionusing electrochemical probe electrode(s) 7 d. The reaction zone 6 may bethe same composition as the hydrophilic matrix conductor 2, as in theelectrophoretic separation application of this device described later.In other applications of the micro-analytical system, reaction zone 6may be a different composition. For example in the ligand-binding assayapplication of the micro-analytical system reaction zone 6 containsreagents that bind with species being transported along conductor 2.

Referring to the B-B′ cross-section of FIG. 5C, in operation a portionof the top surface of the device is immersed in an aqueous medium. Water50 is transported as vapor through the water vapor-permeable cover layer3 into the initially dry, inactive hydrophilic-solid matrix conductors 2and 2 d, reservoirs 2 a, 2 b and 2 c, and reaction or separation zone 6.The cover layer 3 is otherwise insulating, that is, it does nottransport other solute species, ions or electrons.

One use of the micro-analytical system of FIG.5 is in a ligand-bindingassay. In this application an electrolyte solution containing the targetmolecule to be assayed is introduced into the hydrophilic-matrixconductor 2 through orifice 4. This is achieved by electrokineticpumping when a positive voltage is applied to the electrolyte solutionby integral electrode 7 e relative to the voltage of waste reservoir 2 bapplied through electrode 7 b. Thus, sample solution is pumped through 2to zone 6 which contains an immobilized receptor or capture module thatbinds the target molecule. The bound target molecule may be detectedwithin zone 6. A typical detection strategy known in the art is tointroduce a label molecule that also binds to the target molecule. Thelabel molecule will traverse the conductor 2 to zone 6 where it binds tothe target molecule. The concentration of the bound entity including thebound target molecule and label is detected by assaying theconcentration of the label as is known in the art. Labels may includebut are not limited to fluorescent, luminescent, optically absorbing orenzymatic labels. In this assay method only the bound labelconcentration that is related to bound target molecule concentration ismeasured, because unbound label molecules are removed from the reactionzone 6 by electrokinetic pumping and are not detected. The concentrationof chemical species within the reaction layer 6 is measured by opticaldetector 11 or by integral electrochemical probe electrode 7 d. Inoptical detection methods detectors are usually off-chip devices,although integral optical detection devices are known in the art ofligand-binding devices. Electrochemical detectors are particularlysuited to integration as described in this embodiment of the invention.An integral electrochemical probe electrode 7 d (or array of electrodesconstituting an electrochemical cell) and the associated isolationinsulator 8 d are shown in FIG. 5A. A passage 9 d that connects probeelectrode 7 d to the reaction zone 6 is shown in FIG. 5B and 5D. Alsoshown are one or more electrode coatings 10 interposed between the probeelectrode and the reaction zone. In this configuration, theelectrochemical probe electrode(s) 7 d and coatings 10 togetherconstitute a biosensor detector located in a zone of thehydrophilic-matrix conductor.

There are numerous combinations of electrodes 7 d and coatings 10 knownin the prior art of electrochemical biosensors. Indeed, some biosensorshave been used in prior-art separation devices using electrochemicaldetection.

To illustrate the types of biosensors that might be used in a device ofthe present invention, consider a reaction zone 6 in which a reactiontakes place that produces a change of pH. Electrodes 7 d and coating 10might then be a pH electrode with a pH selective membrane. In anotherexample, the reaction being probed might produce a change in hydrogenperoxide concentration for example if the label molecule is the enzymeglucose oxidase. In this example the electrode 7 d is a platinum metalanode and the coating 10 is a hydrogen peroxide selective layer. Thoseskilled in the art will appreciate that there are many possiblebiosensor devices that could be used in this invention.

In those cases where the probe reaction is enzyme based, such as in aligand binding assay using an enzyme probe, it can be advantageous tointroduce substrate for the enzyme reaction after the binding reactionhas occurred. This can be achieved by electrokinetic pumping of theenzyme substrate contained in reagent reservoir 2 c. Reagent flows from2 c via hydrophilic-matrix conductors 2 d and 2 through reaction zone 6to waste reservoir 2 b. Pumping is achieved by applying a positivevoltage to reagent reservoir 2 c through electrode 7 c relative to thevoltage of waste reservoir 2 b. Those skilled in the art ofelectrochemical detection in electrostatically pumped systems appreciatethat there are other arrangements of reaction zone 6 and itselectrochemical detectors with respect to the high voltageelectrokinetic pumping electrodes. For example when pumping electrode 7b is located just upstream of 6 in conductor 2 electrode 7 d is locatedoutside of the high field region, thus simplifying the electrochemicaldetection process. Such other electrode arrangements are clearlycontemplated as variations of the current invention.

Another application of the micro-analytical system of FIG. 5A is inelectrostatically pumped separations. In this case, the reaction zone 6is a separation column. Its contains a matrix suitable for separatingspecies transported through it. In use, a carrier electrolyte is pumpedfrom carrier reservoir 2 a to waste reservoir 2 b. Pumping is achievedby applying a positive voltage to carrier reservoir 2 a throughelectrode 7 a relative to waste reservoir 2 b. A segment of sample iselectrostatically pumped through port 4 into the carrier electrolyteflowing in conductor 2. This is achieved by switching the positivepotential from carrier reservoir 2 a applied through electrode 7 a tothe sample solution applied through electrode 7 e contacting it, thenback to 7 a. Species become spatially separated as they areelectrostatically transported along the hydrophilic-matrix in theseparation region 6 as powered by electrodes 7 a and 7 b. Chargedspecies are separated by differential electrical mobility(electrophoresis), uncharged species are separated by differentialresidence on absorbing surfaces of the separation medium within thehydrophilic matrix (chromatography). The concentration versus timeprofile of separated chemical species as they traverse the reaction zone6 is monitored by an optical detector 11 or an integral electrochemicalbiosensor (electrode 7 d with coatings 10). The composition ofseparation zone 6 could be the same as hydrophilic-matrix conductor 2 inthe simplest implementation of the device in an electrophoreticseparation. In another implementation in a chromatographic separation,zone 6 may be two components as described in the hydrophilic-matrixconductor of FIG. 3E.

In one specific example of a separation application, the species to beseparated are DNA fragments with fluorescent labels, such as might beobtained from a Sanger reaction or a primer extension reaction.

In one method of manufacture of the micro-analytical system of the FIG.5A embodiment, a single layer of a hydrophilic-matrix material isdeposited onto a planar insulating substrate with integral thin filmelectrodes. The hydrophilic matrix is formed into the pattern of theintegrated-electrokinetic circuit consisting of reservoirs andconductors shown in FIG. 5A using photo-processing methods describedearlier. Either a single circuit or an array of circuits on a chip canbe fabricated with the same photo-process. Reagents are added tophoto-formed reservoirs by an impregnation step using a solution thatcontains reagent applied locally over a reservoir region. The solutionis applied by a process of dispensing from a nozzle, spotting or anink-jet deposition process. Different reservoir contents can be achievedby applying different impregnating solutions over each reservoir. Coverlayer 3 is then deposited and formed using processes described earlier.

The micro-analytical system of FIG. 5 is convenient to package becauseall of the component reservoirs and conductors are electrically isolatedfrom the sample fluid and from one another by the cover layer 3.However, the reservoirs are not vented in this device, they are sealed.In this case there will be an internal pressure build-up within thesealed reservoirs resulting from significant efflux or influx ofelectrolyte during pumping, such back-pressure impeding further pumping.Thus this design is appropriate when the amount of material pumped intoor out of a reservoir is small compared with its volume. Clearly it isalso possible to provide vent openings through cover layer 3 over eachreservoir 2 a, 2 b and 2 c to connect them to external electrolytesolution reservoirs in applications where the amount of material to bepumped is sufficient to cause build-up of back-pressure in a sealedreservoir system. In such a vented reservoir device packaging is morecomplex since the electrical isolation of external reservoirs isnecessary to operate the various pumping functions as is appreciated bythose in the art.

With the invented micro-analytical system using the inventedintegrated-electrokinetic circuits, it is now possible to perform manydifferent micro-analytical procedures on a chip. Thus, the use of theinvented devices is not limited to ligand-binding assays and separationsthat described in the embodiment of FIG. 5. It is contemplated thatdifferent arrangements of integrated-electrokinetic circuit componentsaccording to this invention can provide micro-analytical systems toperform numerous analytical functions. Some otherintegrated-electrokinetic circuits and components according to thisinvention used in additional analytical applications include but are notlimited to: devices with reaction zones incorporating pcr reactions,devices with reaction zones supporting primer extension reactions ingeneral, devices with reaction zones incorporating restriction enzymes;integrated devices combining the above reaction zones with ahydrophilic-matrix separation column to analyze reaction components,

FIG. 6 shows another embodiment of the invention. This device isconfigured as an array of electrokinetic pumps for transport of a samplesolution through an array of reaction zones. In this device ahydrophilic-matrix conductor 2 is deposited onto a planar insulatingsubstrate with integral electrodes 7 a and 7 b. The hydrophilic matrixis formed as a parallel array of branch elements joining at a commonreservoir 2 b. Conductors and reservoir are coated with a cover layer 3of a water vapor-permeable insulator. The device has an array ofopenings 4 for influx of a sample solution into the hydrophilic-matrixconductor 2. There is one opening 4 in each of the conductor branches. Areaction zone 6 is provided at each opening.

In a specific example of the device of FIG. 6 the reaction zones 6contain immobilized binding molecules. Thus, this embodiment nowprovides ligand binding arrays and their processes for the manufacture.Such devices are very familiar in the fields of immunoassay and DNAhybridization probes with the added benefit that each element of thebinding array can have the sample solution electrostatically pumped toit under device control. In operation, a portion of the top surface ofthe device is immersed in a sample solution containing one or morespecies for assay. As in the previous embodiments of this invention,water 50 is transported as its vapor into the hydrophilic matrix. Oncewet-up the activated hydrophilic matrix becomes a conductingelectrolyte. Electrokinetic transport occurs when a voltage is appliedto the electrodes 7 a and 7 b. In this embodiment it is preferable thatthe hydrophilic matrix be chosen with a large electroosmotic coefficientsuch that electroosmosis of the entire solution is the fastest transportmode. The purpose of the electrokinetic pump is to transport targetmolecules from the bulk sample solution to the reaction zone containingligand where they are bound and detected. Electrokinetic transport ofsample solution containing target molecules to the binding site enhancesthe speed and sensitivity of the ligand-binding reaction compared to thestandard ligand-binding array where the target molecules diffuse fromthe bulk solution to the binding site.

Each of the reaction zones 6 of the FIG. 6 embodiment can contain adifferent binding molecule as is typical of ligand-binding arrays. In avariation of the FIG. 6 embodiment it is also possible to configure aseparate electrode for each of the branch elements of the hydrophilicmatrix 2. In this way there is additional flexibility to apply differentvoltages for each branch, or to regulate the timing at which each pumpis activated. Those skilled in the art will recognize that there can bedifferent arrangements of the location of pumping electrodes relative toopenings 4 and reaction zones 6 that will also achieve the desiredobject of electrostatically pumping a test solution through the reactionzone. For example the opening could be located over a hydrophilic matrixconductor between the integral pumping electrodes. Also, one of theelectrokinetic-pump electrodes could be immersed in, and in contact withthe sample solution. Non-integral electrodes immersed in aqueousreservoirs connected to the sample solution and reservoir 2 b also couldprovide the electrokinetic pump's power. The detailed position of theligand-binding elements 6 relative to the orifices 4 and conductor 2maybe somewhat different from the schematic of FIG. 6. Orifices 4 may belocated at an end location of the conduit as in the embodiment ofFIG. 1. Instead of locating the ligand-binding elements as coatings overorifices 4 as shown in FIG. 6, the ligand-binding elements may beco-planar with the conductor 2.

In the example shown in FIG. 6 integral electrodes 7 a and 7 b,hydrophilic-matrix conductors 2 and water-permeable insulator layers 3with openings 4 are fabricated on a planar insulator 1, as in theprevious embodiments of the invention. The ligand-binding elements maybe applied onto openings 4 by dispensing from a nozzle, by ink-jetprinting, or by a spotting processes as are known in the art ofligand-binding arrays.

Although the invention has been described above with reference tospecific preferred embodiments and examples of the device and method ofmanufacture of the invention, it will be understood that other specificdevices and methods are also encompassed by the present invention whichis only defined by the scope of the appended claims.

1. A device for electrokinetic transport of an aqueous solute,comprising an electrically insulating substrate; a conductor element forelectrokinetic transport of the solute, the conductor element being inthe form of a solid hydrophilic-matrix layer on the substrate, thematrix layer being in a substantially dry, inactive state whereinelectrokinetic transport is substantially prevented; a pair of spacedapart electrodes in electric contact with the conductor element atspaced apart locations for applying an electric potential across theconductor element; and a cover layer for electrically insulating andencapsulating the conductor element on the substrate, the cover layerbeing made of an encapsulating material which is water vapour permeable,but impermeable to the solute; whereby exposure of the hydrophilicmatrix to water converts the matrix from the inactive state to ahydrated, active state permitting electrokinetic transport of thesolute.
 2. The device of claim 1, further comprising a means forintroducing water into the conductor element.
 3. The device of claim 2,further comprising a means for introducing the aqueous solute into theconductor element.
 4. The device of claim 1, wherein the solidhydrophilic-matrix layer is micro-fabricated onto the substrate.
 5. Thedevice of claim 1, wherein the solid hydrophilic-matrix layer is a dryreagent film.
 6. The device of claim 1, wherein the cover layer ismicro-fabricated onto the conductor element and the substrate.
 7. Thedevice of claim 1, wherein at least one of the cover layer and thesubstrate has at least one portion which is permeable to water vapor. 8.The device of claim 1, wherein the substrate consists of electricallyinsulating material.
 9. The device of claim 1, wherein the substrateincludes a layer of electrically conductive material and a layer ofelectrically insulating material intermediate the layer of conductivematerial and the conductor element.
 10. The device of claim 1, whereinone of the substrate and the cover layer has a pair of openings forinput and output of the solute species to be transported through theconductor element.
 11. The device of claim 1, comprising at least oneregion for introducing the solute for one of transport, separation andchemical reaction within the conductor element, and a separate means forintroducing water only into the conductor element.
 12. A device forelectrokinetic transport of an aqueous solute, comprising anelectrically insulating substrate; a conductor element forelectrokinetic transport of the solute, the conductor element being inthe form of a solid hydrophilic-matrix layer on the substrate, thematrix layer being in a substantially dry, inactive state whereinelectrokinetic transport is substantially prevented and having a firstsurface engaging the substrate and a second surface; a cover layer forelectrically insulating and covering the second surface, the cover layerbeing impermeable to the solute; and a pair of spaced apart electrodesin electric contact with the conductor element at spaced apart locationsfor applying an electric potential across the conductor element; wherebyexposure of the hydrophilic matrix to water converts the matrix from theinactive state to a hydrated, active state permitting electrokinetictransport of the solute and wherein the electrodes are applied to thesubstrate and the device further includes an insulator layer forelectrically insulating each electrode, the insulator layer having anopening in each region of overlap between one of the electrodes and theconductor element for permitting electric contact of the conductorelement with the integral electrodes for electrokinetic pumping.
 13. Adevice for electrokinetic transport of an aqueous solute, comprising anelectrically insulating substrate; a conductor element forelectrokinetic transport of the solute, the conductor element being inthe form of a solid hydrophilic-matrix layer on the substrate, thematrix layer being in a substantially dry, inactive state whereinelectrokinetic transport is substantially prevented and having a firstsurface engaging the substrate and a second surface; a cover layer forelectrically insulating and covering the second surface, the cover layerbeing impermeable to the solute; and a pair of spaced apart electrodesin electric contact with the conductor element at spaced apart locationsfor applying an electric potential across the conductor element; wherebyexposure of the hydrophilic matrix to water converts the matrix from theinactive state to a hydrated, active state permitting electrokinetictransport of the solute and the device further comprising an inputregion for supply of solute into the conductor element and an outputregion spaced apart therefrom for removal of transported solute from theconductor element.
 14. The device of claim 13, wherein the hydrophilicmatrix of the conductor element is water insoluble in the input andoutput regions.
 15. The device of claim 14, wherein the conductorelement further comprises a reservoir region intermediate the input andoutput regions and including at least one chemical reactant forinteraction with the transported solute.
 16. The device of claim 15,further comprising an electrode for applying an electric potential tothe reservoir region.
 17. The device of claim 15, wherein the conductorelement includes a plurality of the reservoir regions.